Treatment of acute exacerbations of chronic obstructive pulmonary disease by antagonism of the il-20r

ABSTRACT

The present invention relates to methods and pharmaceutical compositions for the treatment of acute exacerbation of chronic obstructive pulmonary disease. In particular, the present invention relates to a method of treating acute exacerbation of chronic obstructive pulmonary disease in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an antagonist of IL-20 cytokines.

The present invention relates to methods and pharmaceutical compositionsfor the treatment of acute exacerbation of chronic obstructive pulmonarydisease.

BACKGROUND OF THE INVENTION

Chronic obstructive pulmonary disease (COPD) represents a severe andincreasing global health problem. By 2020, COPD will have increased from6th (as it is currently) to the 3rd most common cause of deathworldwide. In the United States, COPD is believed to account for up to120,000 deaths per year. The clinical course of COPD is characterized bychronic disability, with intermittent, acute exacerbations which may betriggered by a variety of stimuli including exposure to pathogens,inhaled irritants (e.g., cigarette smoke), allergens, or pollutants.“Acute exacerbation” refers to worsening of a subject's COPD symptomsfrom his or her usual state that is beyond normal day-to-day variations,and is acute in onset. Acute exacerbations of COPD greatly affect thehealth and quality of life of subjects with COPD. Acute exacerbation ofCOPD is a key driver of the associated substantial socioeconomic costsof the disease. Multiple studies have also shown that prior exacerbationis an independent risk factor for future hospitalization for COPD. Inconclusion, exacerbations of COPD are of major importance in terms oftheir prolonged detrimental effect on subjects, the acceleration indisease progression and the high healthcare costs. However up to now,there is no method for the treatment of acute exacerbation of COPD.

Based on their genes and protein structures, IL-19, IL-20 and IL-24 forma subgroup in the IL-10 cytokine family: the IL-20 cytokines that arevery close to IL-22. IL-19 and IL-20 act via a receptor complex thatconsists of the IL-20R1 and IL-20R2 chains present on epithelial andantigen-presenting cells. IL-20 and IL-24 are additionally able tosignal via a second receptor complex (IL-22R1/IL-20R2). Recent reportshave shown that IL-20 cytokines function as proinflammatory cytokinesthat are involved in inflammatory diseases, such as psoriasis,rheumatoid arthritis, atherosclerosis, ischemic stroke, and renalfailure. This cytokine family affects the growth and differentiation ofepithelial cells but also induce a number of chemokines, anti-microbialpeptides and growth factors. Given their origins and their properties,these cytokines probably control the cross-talk between immune andresident cells during mucosal immunity and wound healing. However therole of IL-20 cytokines has never been investigated in acuteexacerbation of COPD.

SUMMARY OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor the treatment of acute exacerbation of chronic obstructive pulmonarydisease. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

Bacterial infections are a major cause of disease exacerbation in lunginflammatory disorders including chronic obstructive pulmonary disease(COPD). In this disease, bacterial susceptibility is related to a defectin Th17 cytokines whereas IL-20 cytokines are overexpressed in the lung.In parallel, an immunosuppressive role for IL-20 cytokines has beenidentified during infection that can be therapeutically targeted toalter susceptibility to infection.

Accordingly, the present invention relates to a method of treating acuteexacerbation of chronic obstructive pulmonary disease in a subject inneed thereof comprising administering the subject with a therapeuticallyeffective amount of an antagonist of IL-20 cytokines.

As used herein the term “acute exacerbation” has its general meaning inthe art and refers to worsening of a subject's COPD symptoms from his orher usual state that is beyond normal day-to-day variations, and isacute in onset. Typically, the acute exacerbation of COPD is manifestedby one or more symptoms selected from worsening dyspnea, increasedsputum production, increased sputum purulence, change in color ofsputum, increased coughing, upper airway symptoms including colds andsore throats, increased wheezing, chest tightness, reduced exercisetolerance, fatigue, fluid retention, and acute confusion, and saidmethod comprises reducing the frequency, severity or duration of one ormore of said symptoms. Acute exacerbation may have various etiologies,but typically may be caused by viral infections, bacterial infections,or air pollution. For example, approximately 50% of acute exacerbationsare due primarily to the bacteria Streptococcus pneumoniae, Haemophilusinfluenzae, and Moraxella catarrhalis (all of them causing pneumonia).Viral pathogens associated with acute exacerbations in subjects withCOPD include rhinoviruses, influenza, parainfluenza, coronavirus,adenovirus, and respiratory syncytial virus.

In some embodiments, the acute exacerbation of COPD is caused by abacterial infection. In some embodiments, the acute exacerbation of COPDis caused by a viral infection. In some embodiments, the acuteexacerbation of COPD is caused by air pollution.

In some embodiments, the subject experienced an acute exacerbation ofCOPD or is at risk of experiencing an acute exacerbation of COPD. Insome embodiments, the subject has experienced at least one acuteexacerbation of COPD in the past 24 months. In one particularembodiment, the subject has experienced at least one acute exacerbationof COPD in the past 12 months. In some embodiments, subject is afrequent exacerbator. As used herein the term “frequent exacerbator”refers to a subject who suffers from or is undergoing treatment for COPDand who experiences at least 2, and more typically 3 or more, acuteexacerbations during a 12 month period.

In some embodiments, “treating” refers to treating an acute exacerbationof COPD, reducing the frequency, duration or severity of an acuteexacerbation of COPD, treating one or more symptoms of acuteexacerbation of COPD, reducing the frequency, duration or severity ofone or more symptoms of an acute exacerbation of COPD, preventing theincidence of acute exacerbation of COPD, or preventing the incidence ofone or more symptoms of acute exacerbation of COPD, in a human. Thereduction in frequency, duration or severity is relative to thefrequency, duration or severity of an acute exacerbation or symptom inthe same human not undergoing treatment according to the methods of thepresent invention. A reduction in frequency, duration or severity ofacute exacerbation or one or more symptoms of acute exacerbation may bemeasured by clinical observation by an ordinarily skilled clinician withexperience of treating COPD subjects or by subjective self evaluationsby the subject undergoing treatment. Clinical observations by anordinarily skilled clinician may include objective measures of lungfunction, as well as the frequency with which intervention is requiredto maintain the subject in his or her most stable condition, and thefrequency of hospital admission and length of hospital stay required tomaintain the subject in his or her most stable condition. Typically,subjective self evaluations by a subject are collected usingindustry-recognized and/or FDA-recognized subject reported outcome (PRO)tools. Such tools may allow the subject to evaluate specific symptoms orother subjective measures of quality of life. An example of one subjectreported outcome tool is Exacerbations from Pulmonary Disease Tool(EXACT-PRO), which is currently being developed for evaluating clinicalresponse in acute bacterial exacerbations by United BioSourceCorporation along with a consortium of pharmaceutical industry sponsorsin consultation with the FDA.

In some embodiments, the treatment is a prophylactic treatment. As usedherein, the term “prophylactic treatment” refer to any medical or publichealth procedure whose purpose is to prevent a disease. As used herein,the terms “prevent”, “prevention” and “preventing” refer to thereduction in the risk of acquiring or developing a given condition, orthe reduction or inhibition of the recurrence or said condition in asubject who is not ill, but who has been or may be near a subject withthe disease.

As used herein, the term “IL-20 cytokines” has its general meaning inthe art and refers to a subgroup in the IL-10 cytokine family whichcomprises IL-19 (Exemplary Human NCBI Reference Sequence: NP_715639.1),IL-20 (Exemplary Human NCBI Reference Sequence: NP_061194.2) and IL-24(Exemplary Human NCBI Reference Sequence: NP_006841.1). IL-20 cytokinesare very close to IL-22. IL-19 and IL-20 act via a receptor complex thatconsists of the IL-20R1 and IL-20R2 chains present on epithelial andantigen-presenting cells. IL-20 and IL-24 are additionally able tosignal via a second receptor complex (IL-22R1/IL-20R2). Examples ofhuman receptors for IL-20 cytokines include hIL-20R1 (also known asCRF2-8; IL-20RA; IL-20R-alpha) (Exemplary Human NCBI Reference Sequence:NP_055247.3), hIL-20R2 (also known as IL-20RB; IL-20R-beta) (ExemplaryHuman NCBI Reference Sequence: NP_653318.2) and hIL-22R1 (Exemplary NCBIReference Sequence: NP_067081.2). More particularly, sequences of humanreceptors for IL-20 cytokines have been described; for example, in U.S.Pat. Nos. 6,610,286; 7,122,632; 7,393,684; and 7,537,761; and U.S. Pat.App. Pub. Nos. 2006/0263850 A1; 2006/0263851 A1; 2008/0247945 A1, and2009/0074661 A1.

The “antagonist of IL-20 cytokines” to be used in the methods describedherein is a molecule that blocks, suppresses, or reduces (includingsignificantly) the biological activity of IL-20 cytokines, includingdownstream pathways mediated by signaling of IL-20 cytokines, such asreceptor binding and/or elicitation of a cellular response to IL-20cytokines. Thus the term “antagonist of IL-20 cytokines” implies nospecific mechanism of biological action whatsoever, and is deemed toexpressly include and encompass all possible pharmacological,physiological, and biochemical interactions with IL-20 cytokines andreceptors whether direct or indirect. For purpose of the presentdisclosure, it will be explicitly understood that the term “antagonistof IL-20 cytokines” encompass all the previously identified terms,titles, and functional states and characteristics whereby the IL-20cytokines themselves, a biological activity of IL-20 cytokines(including but not limited to its ability to control expression of IL-17and IL-22 cytokines during acute exacerbation of COPD), or theconsequences of the biological activity, are substantially nullified,decreased, or neutralized in any meaningful degree, e.g., by at least20%, 50%, 70%, 85%, 90%, 100%, 150%, 200%, 300%, or 500%, or by 10-fold,20-fold, 50-fold, 100-fold, 1000-fold, or 10⁴-fold.

Exemplary antagonists of IL-20 cytokines include, but are not limitedto, an antibody directed against a IL-20 cytokine, an anti-sense nucleicacid molecule directed to an IL-20 cytokine (including an anti-sensenucleic acid directed to a nucleic acid encoding IL-20), a smallinterfering RNA (siRNA) directed toward an nucleic acid encoding for aIL-20 cytokine, a microRNA directed toward a nucleic acid encoding for aIL-20 cytokine, an anti-antibody directed against a receptor of a IL-20cytokine (e.g., an antibody specifically binds IL-20R1, IL-20R2, IL-22RIor the dimeric complex formed thereby), an antisense nucleic acidmolecule directed to a subunit of a receptor for a IL-20 cytokine, ansiRNA or a microRNA directed to a nucleic acid encoding for a subunit ofa receptor for a IL-20 cytokine. In some embodiments, an antagonist ofIL-20 cytokines binds to an IL-20 cytokine or a receptor and preventsthe formation of the complex between the receptor and the cytokine,thereby inhibiting the signaling pathway. In some embodiments, anantagonist of IL-20 cytokines inhibits or reduces synthesis and/orproduction (release) of an IL-20 cytokine.

In some embodiments, the antagonist of IL-20 cytokines of the presentinvention is not an antibody directed against the IL-22R1 receptor, ananti-sense nucleic acid molecule directed toward an nucleic acidencoding for the IL-22R1, a small interfering RNA (siRNA) directedtoward an nucleic acid encoding for a IL-22R1 receptor, a microRNAdirected toward a nucleic acid encoding for a IL-22R1 receptor. In thismanner, the signaling pathway of IL-22 is not disturbed.

In some embodiments, the antagonist of IL-20 cytokines is an antibody.For instance, the antibody disclosed herein specifically binds a targetantigen, such as human IL-20 cytokine or one of the subunits of a humanreceptor for an IL-20 cytokine (e.g., IL-20R1). In some embodiments, theantagonist of IL-20 cytokines is selected from the group consisting ofanti-IL-19 antibodies, anti-IL-20 antibodies, anti-IL-24 antibodies,anti-IL20R1 antibodies, and anti-IL20R2 antibodies.

An antibody that “specifically binds” (used interchangeably herein) to atarget or an epitope is a term well understood in the art, and methodsto determine such specific binding are also well known in the art. Amolecule is said to exhibit “specific binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular target antigen than it does withalternative targets. An antibody “specifically binds” to a targetantigen if it binds with greater affinity, avidity, more readily, and/orwith greater duration than it binds to other substances. For example, anantibody that specifically (or preferentially) binds to an epitope is anantibody that binds this epitope with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other epitopespresent in an IL-20 cytokine (or its receptor subunit) or epitopes thatare not present in an IL-20 cytokine (or its receptor subunit). It isalso understood by reading this definition that, for example, anantibody that specifically binds to a first target antigen may or maynot specifically or preferentially bind to a second target antigen. Assuch, “specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

The term “antibody” is thus used to refer to any antibody-like moleculethat has an antigen binding region, and this term includes antibodyfragments that comprise an antigen binding domain such as Fab′, Fab,F(ab′)₂, single domain antibodies (DABs), TandAbs dimer, Fv, scFv(single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies,diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fabfusions, bispecific or trispecific, respectively); sc-diabody;kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager,scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domainantibody, bispecific format); SIP (small immunoprotein, a kind ofminibody); SMIP (“small modular immunopharmaceutical” scFv-Fc dimer;DART (ds-stabilized diabody “Dual Affinity ReTargeting”); small antibodymimetics comprising one or more CDRs and the like. The techniques forpreparing and using various antibody-based constructs and fragments arewell known in the art (see Kabat et al., 1991, specifically incorporatedherein by reference). Diabodies, in particular, are further described inEP 404, 097 and WO 93/1 1 161; whereas linear antibodies are furtherdescribed in Zapata et al. (1995). Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also besynthesized by recombinant techniques or can be chemically synthesized.Techniques for producing antibody fragments are well known and describedin the art. For example, each of Beckman et al., 2006; Holliger &Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al.,1996; and Young et al., 1995 further describe and enable the productionof effective antibody fragments.

In natural antibodies, two heavy chains are linked to each other bydisulfide bonds and each heavy chain is linked to a light chain by adisulfide bond. There are two types of light chain, lambda (l) and kappa(k). There are five main heavy chain classes (or isotypes) whichdetermine the functional activity of an antibody molecule: IgM, IgD,IgG, IgA and IgE. Each chain contains distinct sequence domains. Thelight chain includes two domains, a variable domain (VL) and a constantdomain (CL). The heavy chain includes four domains, a variable domain(VH) and three constant domains (CH1, CH2 and CH3, collectively referredto as CH). The variable regions of both light (VL) and heavy (VH) chainsdetermine binding recognition and specificity to the antigen. Theconstant region domains of the light (CL) and heavy (CH) chains conferimportant biological properties such as antibody chain association,secretion, trans-placental mobility, complement binding, and binding toFc receptors (FcR). The Fv fragment is the N-terminal part of the Fabfragment of an immunoglobulin and consists of the variable portions ofone light chain and one heavy chain. The specificity of the antibodyresides in the structural complementarity between the antibody combiningsite and the antigenic determinant. Antibody combining sites are made upof residues that are primarily from the hypervariable or complementaritydetermining regions (CDRs). Occasionally, residues from nonhypervariableor framework regions (FR) influence the overall domain structure andhence the combining site. Complementarity Determining Regions or CDRsrefer to amino acid sequences which together define the binding affinityand specificity of the natural Fv region of a native immunoglobulinbinding site. The light and heavy chains of an immunoglobulin each havethree CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2,H-CDR3, respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. Framework Regions (FRs) refer to amino acid sequences interposedbetween CDRs. In addition, determination of CDR regions in an antibodyis well within the skill of the art. There are at least two techniquesfor determining CDRs: (1) an approach based on cross-species sequencevariability (i.e., Kabat et al. Sequences of Proteins of ImmunologicalInterest, (5th ed., 1991, National Institutes of Health, Bethesda Md.));and (2) an approach based on crystallographic studies ofantigen-antibody complexes (Chothia et al. (1989) Nature 342:877;Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). As used herein,a CDR may refer to CDRs defined by either approach or by a combinationof both approaches.

The term “Fab” denotes an antibody fragment having a molecular weight ofabout 50,000 and antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating IgG with a protease, papaine, are bound togetherthrough a disulfide bond.

The term “F(ab′)2” refers to an antibody fragment having a molecularweight of about 100,000 and antigen binding activity, which is slightlylarger than the Fab bound via a disulfide bond of the hinge region,among fragments obtained by treating IgG with a protease, pepsin.

The term “Fab′” refers to an antibody fragment having a molecular weightof about 50,000 and antigen binding activity, which is obtained bycutting a disulfide bond of the hinge region of the F(ab′)2.

A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VLheterodimer which is usually expressed from a gene fusion including VHand VL encoding genes linked by a peptide-encoding linker. “dsFv” is aVH::VL heterodimer stabilised by a disulfide bond. Divalent andmultivalent antibody fragments can form either spontaneously byassociation of monovalent scFvs, or can be generated by couplingmonovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

Monoclonal antibodies may be generated using the method of Kohler andMilstein (Nature, 256:495, 1975). To prepare monoclonal antibodiesuseful in the invention, a mouse or other appropriate host animal isimmunized at suitable intervals (e.g., twice-weekly, weekly,twice-monthly or monthly) with the relevant antigenic forms (e.g. anIL-20 cytokine or a receptor). The animal may be administered a final“boost” of antigen within one week of sacrifice. It is often desirableto use an immunologic adjuvant during immunization. Suitable immunologicadjuvants include Freund's complete adjuvant, Freund's incompleteadjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants suchas QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides.Other suitable adjuvants are well-known in the field. The animals may beimmunized by subcutaneous, intraperitoneal, intramuscular, intravenous,intranasal or other routes. A given animal may be immunized withmultiple forms of the antigen by multiple routes.

Briefly, the recombinant antigen (e.g. an IL-20 cytokine or a receptor)may be provided by expression with recombinant cell lines. For instancereceptors (e.g. IL-20R1 or IL-20R2) may be provided in the form of humancells expressing the receptor at their surface. Recombinant forms of thecytokine or receptor may be provided using any previously describedmethod. Following the immunization regimen, lymphocytes are isolatedfrom the spleen, lymph node or other organ of the animal and fused witha suitable myeloma cell line using an agent such as polyethylene glycolto form a hydridoma. Following fusion, cells are placed in mediapermissive for growth of hybridomas but not the fusion partners usingstandard methods, as described (Coding, Monoclonal Antibodies:Principles and Practice: Production and Application of MonoclonalAntibodies in Cell Biology, Biochemistry and Immunology, 3rd edition,Academic Press, New York, 1996). Following culture of the hybridomas,cell supernatants are analyzed for the presence of antibodies of thedesired specificity, i.e., that selectively bind the antigen. Suitableanalytical techniques include ELISA, flow cytometry,immunoprecipitation, and western blotting. Other screening techniquesare well-known in the field. Preferred techniques are those that confirmbinding of antibodies to conformationally intact, natively foldedantigen, such as non-denaturing ELISA, flow cytometry, andimmunoprecipitation.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The Fc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)2 fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDRS). The CDRs, andin particular the CDRS regions, and more particularly the heavy chainCDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of amammalian antibody may be replaced with similar regions of specific orheterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody.

In some embodiments, the antibody is a humanized antibody. As usedherein, “humanized” describes antibodies wherein some, most or all ofthe amino acids outside the CDR regions are replaced with correspondingamino acids derived from human immunoglobulin molecules. Methods ofhumanization include, but are not limited to, those described in U.S.Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and5,859,205, which are hereby incorporated by reference. The above U.S.Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose fourpossible criteria which may used in designing the humanized antibodies.The first proposal was that for an acceptor, use a framework from aparticular human immunoglobulin that is unusually homologous to thedonor immunoglobulin to be humanized, or use a consensus framework frommany human antibodies. The second proposal was that if an amino acid inthe framework of the human immunoglobulin is unusual and the donor aminoacid at that position is typical for human sequences, then the donoramino acid rather than the acceptor may be selected. The third proposalwas that in the positions immediately adjacent to the 3 CDRs in thehumanized immunoglobulin chain, the donor amino acid rather than theacceptor amino acid may be selected. The fourth proposal was to use thedonor amino acid reside at the framework positions at which the aminoacid is predicted to have a side chain atom within 3 A of the CDRs in athree dimensional model of the antibody and is predicted to be capableof interacting with the CDRs. The above methods are merely illustrativeof some of the methods that one skilled in the art could employ to makehumanized antibodies. One of ordinary skill in the art will be familiarwith other methods for antibody humanization.

In some embodiments, some, most or all of the amino acids outside theCDR regions have been replaced with amino acids from humanimmunoglobulin molecules but where some, most or all amino acids withinone or more CDR regions are unchanged. Small additions, deletions,insertions, substitutions or modifications of amino acids arepermissible as long as they would not abrogate the ability of theantibody to bind a given antigen. Suitable human immunoglobulinmolecules would include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. A“humanized” antibody retains a similar antigenic specificity as theoriginal antibody. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody may be increasedusing methods of “directed evolution”, as described by Wu et al., J.Mol. Biol. 294:151, 1999, the contents of which are incorporated hereinby reference.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369,5,545,806, 5,545,807, 6,150,584, and references cited therein, thecontents of which are incorporated herein by reference. These animalshave been genetically modified such that there is a functional deletionin the production of endogenous (e.g., murine) antibodies. The animalsare further modified to contain all or a portion of the human germ-lineimmunoglobulin gene locus such that immunization of these animals willresult in the production of fully human antibodies to the antigen ofinterest. Following immunization of these mice (e.g., XenoMouse(Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can beprepared according to standard hybridoma technology. These monoclonalantibodies will have human immunoglobulin amino acid sequences andtherefore will not provoke human anti-mouse antibody (KAMA) responseswhen administered to humans. In vitro methods also exist for producinghuman antibodies. These include phage display technology (U.S. Pat. Nos.5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S.Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents areincorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′) 2 Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)2 fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

The various antibody molecules and fragments may derive from any of thecommonly known immunoglobulin classes, including but not limited to IgA,secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known tothose in the art and include but are not limited to human IgG1, IgG2,IgG3 and IgG4.

The binding affinity of an antibody can be less than any of about 100nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, orabout 50 pM to any of about 2 pM. Binding affinity can be expressed KDor dissociation constant, and an increased binding affinity correspondsto a decreased KD. One way of determining binding affinity of antibodiesis by measuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of an anti-IL-20 Fab fragment of an antibody can be determinedby surface plasmon resonance (BIAcore3000™ surface plasmon resonance(SPR) system, BIAcore, INC, Piscaway N.J.). Kinetic association rates(kon) and dissociation rates (koff) (generally measured at 25° C.) areobtained; and equilibrium dissociation constant (KD) values arecalculated as koff/kon.

Examples of anti-IL-20 antibodies include, but are not limited to, thosedisclosed in U.S. Pat. Nos. 7,435,800; 7,115,714; 7,119,175; 7,151,166;and 7,393,684; and PCT publications WO 2007/081465; WO 99/27103; WO2004/085475; and WO 2005052000. In some embodiments, the anti-IL-20antibody described herein is anti-IL-20 antibody 7E, which refers tomonoclonal antibody mAb 7E and its functional variants. MAb 7E isproduced by the hybridoma cell line deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A. and assigned a deposit number PTA-8687. Thishybridoma cell line will be released to the public irrevocably andwithout restriction/condition upon granting a US patent on thisapplication, and will be maintained in the ATCC for a period of at least30 years from the date of the deposit for the enforceable life of thepatent or for a period of 5 years after the date of the most recent. Afunctional variant (equivalent) of mAb7E has essentially the sameepitope-binding specificity as mAb7E and exhibits at least 20% (e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the activity ofneutralizing a signaling pathway mediated by IL-20 as relative to mAb7E.In some embodiments, a functional variant of mAb7E contains the sameregions/residues responsible for antigen-binding as mAb7E, such as thesame specificity-determining residues in the CDRs or the whole CDRs. Theregions/residues that are responsible for antigen-binding can beidentified from amino acid sequences of the heavy chain/light chainsequences of mAb7GW or mAb51D (shown above) by methods known in the art.See, e.g., www.bioinf.org.uk/abs; Almagro, J. Mol. Recognit. 17:132-143(2004); and Chothia et al., J. Mol. Biol. 227:799-817 (1987).

In some embodiments, the anti-IL20R1 antibody used in the methodsdescribed herein is an antibody having the same heavy chain and lightchain variable regions (VH and VL) as those of monoclonal antibodymAb7GW or mAb51D, the monoclonal antibodies, an antigen-binding fragmentthereof, or a functional equivalent of either mAb7GW or mAb51D disclosedin US2011/0256093, which is herein incorporated by reference in itsentirety. A functional equivalent of mAb7GW or mAb51D has the sameepitope-binding specificity as mAb7GW or mAb51D and exhibits at least20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of theactivity of neutralizing a signaling pathway mediated by IL-20R1 asrelative to mAb7GW or mAb51D. In some embodiments, a functionalequivalent of mAb7GW or mAb51D contains the same regions/residuesresponsible for antigen-binding as mAb7GW or mAb51D, such as the samespecificity-determining residues in the CDRs or the whole CDRs. Theregions/residues that are responsible for antigen-binding can beidentified from amino acid sequences of the heavy chain/light chainsequences of mAb7GW or mAb51D (shown above) by methods known in the art.See, e.g., www.bioinf.org.uk/abs; Almagro, J. Mol. Recognit. 17:132-143(2004); and Chothia et al., J. Mol. Biol. 227:799-817 (1987).

In some embodiments, the antagonist of IL-20 cytokines is polypeptide.In some embodiments, polypeptide comprises a functional equivalent of anIL-20 receptor (i.e. IL-20R1, IL-20R2, and IL-22RI). As used herein, a“functional equivalent of an IL-20 receptor” is a polypeptide which iscapable of binding to an IL-20 cytokine, thereby preventing itsinteraction with an IL-20 receptor. The term “functional equivalent”includes fragments, mutants, and muteins of IL-20 receptor. The term“functionally equivalent” thus includes any equivalent of an IL-20receptor obtained by altering the amino acid sequence, for example byone or more amino acid deletions, substitutions or additions such thatthe protein analogue retains the ability to bind to an IL-20 cytokine.Amino acid substitutions may be made, for example, by point mutation ofthe DNA encoding the amino acid sequence. Functional equivalents includemolecules that bind an IL-20 cytokine and comprise all or a portion ofthe extracellular domains of IL-20 receptor so as to form a solublereceptor that is capable to trap the IL-20 cytokine. Thus the functionalequivalents include soluble forms of the IL-20 receptor. A suitablesoluble form of these proteins, or functional equivalents thereof, mightcomprise, for example, a truncated form of the protein from which thetransmembrane domain has been removed by chemical, proteolytic orrecombinant methods. Typically, the functional equivalent is at least80% homologous to the corresponding protein. In a preferred embodiment,the functional equivalent is at least 90% homologous as assessed by anyconventional analysis algorithm. The term “a functionally equivalentfragment” as used herein also may mean any fragment or assembly offragments of IL-20 receptor that binds to an IL-20 cytokine. Accordinglythe present invention provides a polypeptide capable of inhibitingbinding of an IL-20 receptor to an IL-20 cytokine, which polypeptidecomprises consecutive amino acids having a sequence which corresponds tothe sequence of at least a portion of an extracellular domain of anIL-20 receptor, which portion binds to an IL-20 cytokine. In someembodiments, the polypeptide corresponds to an extracellular domain ofan IL-20 receptor. In some embodiments, the polypeptide does notcomprise a functional equivalent of IL-22RI.

In some embodiments, the polypeptide comprises a functional equivalentof an IL-20 receptor which is fused to an immunoglobulin constant domain(Fc region) to form an immunoadhesin. Immunoadhesins can possess many ofthe valuable chemical and biological properties of human antibodies.Since immunoadhesins can be constructed from a human protein sequencewith a desired specificity linked to an appropriate human immunoglobulinhinge and constant domain (Fc) sequence, the binding specificity ofinterest can be achieved using entirely human components. Suchimmunoadhesins are minimally immunogenic to the patient, and are safefor chronic or repeated use. In some embodiments, the Fc region is anative sequence Fc region. In some embodiments, the Fc region is avariant Fc region. In still another embodiment, the Fc region is afunctional Fc region. As used herein, the term “Fc region” is used todefine a C-terminal region of an immunoglobulin heavy chain, includingnative sequence Fc regions and variant Fc regions. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is usually defined to stretch froman amino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof. The adhesion portion and the immunoglobulinsequence portion of the immunoadhesin may be linked by a minimal linker.The immunoglobulin sequence typically, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG1, IgG2, IgG3or IgG4 subtypes, IgA, IgE, IgD or IgM, but typically IgG1 or IgG3. Insome embodiments, the functional equivalent of the IL-20 receptor andthe immunoglobulin sequence portion of the immunoadhesin are linked by aminimal linker. As used herein, the term “linker” refers to a sequenceof at least one amino acid that links the polypeptide of the inventionand the immunoglobulin sequence portion. Such a linker may be useful toprevent steric hindrances. In some embodiments, the linker has 4; 5; 6;7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25;26; 27; 28; 29; 30 amino acid residues. However, the upper limit is notcritical but is chosen for reasons of convenience regarding e.g.biopharmaceutical production of such polypeptides. The linker sequencemay be a naturally occurring sequence or a non-naturally occurringsequence. If used for therapeutical purposes, the linker is typicallynon-immunogenic in the subject to which the immunoadhesin isadministered. One useful group of linker sequences are linkers derivedfrom the hinge region of heavy chain antibodies as described in WO96/34103 and WO 94/04678. Other examples are poly-alanine linkersequences.

In some embodiments, the polypeptide comprises the extracellular domainof the IL-20R1 polypeptide and the extracellular domain of the IL-20RIIwhich are covalently linked together. In some embodiments oneextracellular domain has a constant region of a heavy chain of animmunoglobulin fused to its carboxy terminus and the other extracellulardomain has a constant light chain of an immunoglobulin (Ig) fused to itscarboxy terminus such that the two polypeptides come together to form asoluble receptor and a disulfide bond is formed between the heavy andthe light Ig chains. In some embodiments, a peptide linker could befused to the two carboxy-termini of the extracellular domains to form acovalently bonded soluble receptor.

The polypeptides of the invention may be produced by any suitable means,as will be apparent to those of skill in the art. In order to producesufficient amounts of IL-20 receptor or functional equivalents thereoffor use in accordance with the present invention, expression mayconveniently be achieved by culturing under appropriate conditionsrecombinant host cells containing the polypeptide of the invention.Typically, the polypeptide is produced by recombinant means, byexpression from an encoding nucleic acid molecule. Systems for cloningand expression of a polypeptide in a variety of different host cells arewell known. When expressed in recombinant form, the polypeptide istypically generated by expression from an encoding nucleic acid in ahost cell. Any host cell may be used, depending upon the individualrequirements of a particular system. Suitable host cells includebacteria mammalian cells, plant cells, yeast and baculovirus systems.Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells. HeLacells, baby hamster kidney cells and many others. Bacteria are alsopreferred hosts for the production of recombinant protein, due to theease with which bacteria may be manipulated and grown. A common,preferred bacterial host is E coli.

In some embodiments, it is contemplated that polypeptides used in thetherapeutic methods of the present invention may be modified in order toimprove their therapeutic efficacy. Such modification of therapeuticcompounds may be used to decrease toxicity, increase circulatory time,or modify biodistribution. For example, the toxicity of potentiallyimportant therapeutic compounds can be decreased significantly bycombination with a variety of drug carrier vehicles that modifybiodistribution. In example adding dipeptides can improve thepenetration of a circulating agent in the eye through the blood retinalbarrier by using endogenous transporters. A strategy for improving drugviability is the utilization of water-soluble polymers. Variouswater-soluble polymers have been shown to modify biodistribution,improve the mode of cellular uptake, change the permeability throughphysiological barriers; and modify the rate of clearance from the body.To achieve either a targeting or sustained-release effect, water-solublepolymers have been synthesized that contain drug moieties as terminalgroups, as part of the backbone, or as pendent groups on the polymerchain. Polyethylene glycol (PEG) has been widely used as a drug carrier,given its high degree of biocompatibility and ease of modification.Attachment to various drugs, proteins, and liposomes has been shown toimprove residence time and decrease toxicity. PEG can be coupled toactive agents through the hydroxyl groups at the ends of the chain andvia other chemical methods; however, PEG itself is limited to at mosttwo active agents per molecule. In a different approach, copolymers ofPEG and amino acids were explored as novel biomaterials which wouldretain the biocompatibility properties of PEG, but which would have theadded advantage of numerous attachment points per molecule (providinggreater drug loading), and which could be synthetically designed to suita variety of applications. Those of skill in the art are aware ofPEGylation techniques for the effective modification of drugs. Forexample, drug delivery polymers that consist of alternating polymers ofPEG and tri-functional monomers such as lysine have been used byVectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons orless) are linked to the a- and e-amino groups of lysine through stableurethane linkages. Such copolymers retain the desirable properties ofPEG, while providing reactive pendent groups (the carboxylic acid groupsof lysine) at strictly controlled and predetermined intervals along thepolymer chain. The reactive pendent groups can be used forderivatization, cross-linking, or conjugation with other molecules.These polymers are useful in producing stable, long-circulatingpro-drugs by varying the molecular weight of the polymer, the molecularweight of the PEG segments, and the cleavable linkage between the drugand the polymer. The molecular weight of the PEG segments affects thespacing of the drug/linking group complex and the amount of drug permolecular weight of conjugate (smaller PEG segments provides greaterdrug loading). In general, increasing the overall molecular weight ofthe block co-polymer conjugate will increase the circulatory half-lifeof the conjugate. Nevertheless, the conjugate must either be readilydegradable or have a molecular weight below the threshold-limitingglomular filtration (e.g., less than 60 kDa). In addition, to thepolymer backbone being important in maintaining circulatory half-life,and biodistribution, linkers may be used to maintain the therapeuticagent in a pro-drug form until released from the backbone polymer by aspecific trigger, typically enzyme activity in the targeted tissue. Forexample, this type of tissue activated drug delivery is particularlyuseful where delivery to a specific site of biodistribution is requiredand the therapeutic agent is released at or near the site of pathology.Linking group libraries for use in activated drug delivery are known tothose of skill in the art and may be based on enzyme kinetics,prevalence of active enzyme, and cleavage specificity of the selecteddisease-specific enzymes. Such linkers may be used in modifying theprotein or fragment of the protein described herein for therapeuticdelivery.

In some embodiments, the antagonist of IL-20 cytokines is an inhibitorof expression. An “inhibitor of expression” refers to a natural orsynthetic compound that has a biological effect to inhibit theexpression of a gene (i.e. IL-19, IL-20, IL-24, IL-20R1 or IL-20R2). Insome embodiments, said inhibitor of gene expression is a siRNA, anantisense oligonucleotide or a ribozyme. For example, anti-senseoligonucleotides, including anti-sense RNA molecules and anti-sense DNAmolecules, would act to directly block the translation of RIP2 mRNA bybinding thereto and thus preventing protein translation or increasingmRNA degradation, thus decreasing the level of an IL-20 cytokine or areceptor subunit thereof, and thus activity, in a cell. For example,antisense oligonucleotides of at least about 15 bases and complementaryto unique regions of the mRNA transcript sequence encoding RIP2 can besynthesized, e.g., by conventional phosphodiester techniques. Methodsfor using antisense techniques for specifically inhibiting geneexpression of genes whose sequence is known are well known in the art(e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323;6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) canalso function as inhibitors of expression for use in the presentinvention. Gene expression can be reduced by contacting a subject orcell with a small double stranded RNA (dsRNA), or a vector or constructcausing the production of a small double stranded RNA, such that geneexpression is specifically inhibited (i.e. RNA interference or RNAi).Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of theinvention may be delivered in vivo alone or in association with avector. In its broadest sense, a “vector” is any vehicle capable offacilitating the transfer of the antisense oligonucleotide, siRNA, shRNAor ribozyme nucleic acid to the cells and typically cells expressingRIP2. Typically, the vector transports the nucleic acid to cells withreduced degradation relative to the extent of degradation that wouldresult in the absence of the vector. In general, the vectors useful inthe invention include, but are not limited to, plasmids, phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of the antisenseoligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viralvectors are a preferred type of vector and include, but are not limitedto nucleic acid sequences from the following viruses: retrovirus, suchas moloney murine leukemia virus, harvey murine sarcoma virus, murinemammary tumor virus, and rous sarcoma virus; adenovirus,adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barrviruses; papilloma viruses; herpes virus; vaccinia virus; polio virus;and RNA virus such as a retrovirus. One can readily employ other vectorsnot named but known to the art.

By a “therapeutically effective amount” is meant a sufficient amount ofthe polypeptide (or the nucleic acid encoding for the polypeptide) toprevent for use in a method for the treatment of acute exacerbation ofCOPD at a reasonable benefit/risk ratio applicable to any medicaltreatment. It will be understood that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular subjectwill depend upon a variety of factors including the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific polypeptide employed; and like factorswell known in the medical arts. For example, it is well known within theskill of the art to start doses of the compound at levels lower thanthose required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.However, the daily dosage of the products may be varied over a widerange from 0.01 to 1,000 mg per adult per day. Typically, thecompositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, 50.0, 100, 250 and 500 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject to be treated. Amedicament typically contains from about 0.01 mg to about 500 mg of theactive ingredient, typically from 1 mg to about 100 mg of the activeingredient. An effective amount of the drug is ordinarily supplied at adosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day,especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically the active ingredient of the present invention (i.e. theantagonist of IL-20 cytokines) is combined with pharmaceuticallyacceptable excipients, and optionally sustained-release matrices, suchas biodegradable polymers, to form pharmaceutical compositions. The term“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin. In the pharmaceutical compositions of thepresent invention, the active ingredients of the invention can beadministered in a unit administration form, as a mixture withconventional pharmaceutical supports. Suitable unit administration formscomprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms.

In some embodiment, the antagonist of IL-20 cytokines is administered tothe subject in combination with an anti-bacterial agent, such asantibiotics or antiviral agents. Suitable antibiotics that could becoadministered in combination with the antagonist include, but are notlimited to, at least one antibiotic selected from the group consistingof: ceftriaxone, cefotaxime, vancomycin, meropenem, cefepime,ceftazidime, cefuroxime, nafcillin, oxacillin, ampicillin, ticarcillin,ticarcillin/clavulinic acid (Timentin), ampicillin/sulbactam (Unasyn),azithromycin, trimethoprim-sulfamethoxazole, clindamycin, ciprofloxacin,levofloxacin, synercid, amoxicillin, amoxicillin/clavulinic acid(Augmentin), cefuroxime, trimethoprim/sulfamethoxazole, azithromycin,clindamycin, dicloxacillin, ciprofloxacin, levofloxacin, cefixime,cefpodoxime, loracarbef, cefadroxil, cefabutin, cefdinir, andcephradine. Example of antiviral agents include but are not limited toacyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir;amantadine, rimantadine; ribavirin; zanamavir and/or oseltamavir; aprotease inhibitor, such as indinavir, nelfinavir, ritonavir and/orsaquinavir; a nucleoside reverse transcriptase inhibitor, such asdidanosine, lamivudine, stavudine, zalcitabine, zidovudine; anon-nucleoside reverse transcriptase inhibitor, such as nevirapine,efavirenz.

Combination treatment may also include respiratory stimulants.Corticosteroids may be beneficial in acute exacerbations of COPD.Examples of corticosteroids that can be used in combination with theantagonist of the present invention are prednisolone,methylprednisolone, dexamethasone, naflocort, deflazacort, halopredoneacetate, budesonide, beclomethasone dipropionate, hydrocortisone,triamcinolone acetonide, fluocinolone acetonide, fluocinonide,clocortolone pivalate, methylprednisolone aceponate, dexamethasonepalmitoate, tipredane, hydrocortisone aceponate, prednicarbate,alclometasone dipropionate, halometasone, methylprednisolonesuleptanate, mometasone furoate, rimexolone, prednisolone farnesylate,ciclesonide, deprodone propionate, fluticasone propionate, halobetasolpropionate, loteprednol etabonate, betamethasone butyrate propionate,flunisolide, prednisone, dexamethasone sodium phosphate, triamcinolone,betamethasone 17-valerate, betamethasone, betamethasone dipropionate,hydrocortisone acetate, hydrocortisone sodium succinate, prednisolonesodium phosphate and hydrocortisone probutate. Particularly preferredcorticosteroids under the present invention are: dexamethasone,budesonide, beclomethasone, triamcinolone, mometasone, ciclesonide,fluticasone, flunisolide, dexamethasone sodium phosphate and estersthereof as well as6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid (S)-fluoromethyl ester. Still more preferred corticosteroids underthe present invention are: budesonide, beclomethasone dipropionate,mometasone furoate, ciclesonide, triamcinolone, triamcinolone acetonide,triamcinolone hexaacetonide and fluticasone propionate optionally in theform of their racemates, their enantiomers, their diastereomers andmixtures thereof, and optionally their pharmacologically-compatible acidaddition salts. Even more preferred are budesonide, beclomethasonedipropionate, mometasone furoate, ciclesonide and fluticasonepropionate. The most preferred corticosteroids of the present inventionare budesonide and beclomethasone dipropionate.

Bronchodilator dosages may be increased during acute exacerbations todecrease acute bronchospasm. Examples of bronchodilators include but arenot limited to β2-agonists (e.g. salbutamol, bitolterol mesylate,formoterol, isoproterenol, levalbuterol, metaproterenol, salmeterol,terbutaline, and fenoterol), anticholinergic (e.g. tiotropium oripratropium), methylxanthined, and phosphodiesterase inhibitors.

In some embodiments, the antagonist of the invention is administered tothe subject in combination with a vaccine which contains an antigen orantigenic composition capable of eliciting an immune response against avirus or a bacterium. Typically, the vaccine composition is used toeliciting an immune response against at least one bacterium selectedfrom the group consisting of Streptococcus pneumoniae, Staphylococcusaureus, Burkholderis ssp., Streptococcus agalactiae, Haemophilusinfluenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae,Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis,Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila,Serratia marcescens, Mycobacterium tuberculosis, Bordetella pertussis.In particular, the vaccine composition is directed against Streptococcuspneumonia or Haemophilus influenza. More particularly, the vaccinecomposition is directed against Non-typeable Haemophilus influenzae(NTHi). Typically, vaccine composition typically contains whole killedor inactivated (eg., attenuated) bacteria isolate(s). However, solubleor particulate antigen comprising or consisting of outer cell membraneand/or surface antigens can be suitable as well, or instead of, wholekilled organisms. In one or more embodiments, the outer cellularmembrane fraction or membrane protein(s) of the selected isolate(s) isused. For instance, NTHi OMP P6 is a highly conserved 16-kDa lipoprotein(Nelson, 1988) which is a target of human bactericidal antibody andinduces protection both in humans and in animal models. In chronicpulmonary obstructive disease (COPD), OMP P6 has been shown to evoke alymphocyte proliferative response that is associated with relativeprotection from NTHi infection (Abe, 2002). Accordingly, OMP P6 or anyother suitable outer membrane NTHi proteins, polypeptides (eg., P2, P4and P26) or antigenic fragments of such proteins or polypeptides canfind application for a NTHi vaccine. Soluble and/or particulate antigencan be prepared by disrupting killed or viable selected isolate(s). Afraction for use in the vaccine can then be prepared by centrifugation,filtration and/or other appropriate techniques known in the art. Anymethod which achieves the required level of cellular disruption can beemployed including sonication or dissolution utilizing appropriatesurfactants and agitation, and combination of such techniques. Whensonication is employed, the isolate can be subjected to a number ofsonication steps in order to obtain the required degree of cellulardisruption or generation of soluble and/or particulate matter of aspecific size or size range. In some embodiments, the vaccinecomposition comprises an adjuvant, in a particular TLR agonist. In oneembodiment, the TLR agonist is selected from the group consisting ofTLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11,TLR12, or TLR13 agonists.

In certain embodiments, oxygen requirements may increase andsupplemental oxygen may be provided.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1—COPD patients have a defective response to Streptococcuspneumoniae (Sp). Production of IL-17 and IL-22 was evaluated by ELISA insupernatants from mononuclear cells from not smoker healthy subjects(controls), smokers healthy subjects and COPD patients. Results wereexpressed as mean±SEM. *: p<0.05 vs controls.

FIG. 2: The expression of IL-19, IL-20 and IL-24 was induced by exposureto cigarette smoke (CS) and by infection with S. pneumoniae (SP) with anadditive effect between both stimuli in the lung of wild type mice.

FIG. 3: The mRNA expression of IL-19 and IL-24 in the human airwayepithelial cells (AEC) BEAS-2B cells, was induced by exposure to CSextract (CSE) but not by infection with S. pneumoniae (SP1). CSE wasadded either at 1/30 or 1/60 dilution (CSE30 vs CSE60).

FIG. 4: The bacterial load was measured in Bronchoalveaolar lavage (BAL)and lung lysates from COPD mice infected with S. pneumoniae (SP1) andtreated with either the isotype control (IgG) or anti-IL-20β antibody.BAL and lungs were collected at day 3 after infection.

FIG. 5: The bacterial load was measured in Bronchoalveaolar lavage (BAL)and lung lysates from COPD mice infected with not typable Haemophilusinfluenza (NTHi) and treated with either the isotype control (IgG) oranti-IL-20β antibody. BAL and lungs were collected at day 1 afterinfection. **: p<0.01 as compared with infected mice exposed to air orto CS (COPD), respectively.

FIG. 6: The secretion of IL-22 was measured in lung cells from COPD miceinfected with not typeable H. influenza (NTHI) and treated with eitherthe isotype control (NTHI+PBS) or anti-IL-20β antibody. Lung cells werecollected either at day 1 and 2 after infection and incubated for 3 daysat 37° C. in medium alone or in presence of heat-killed bacteria (HK).

FIG. 7: Expression of the mRNA encoding for the anti-microbial peptidesS 100A8 and REG3G in the lung tissue from COPD mice infected with S.pneumoniae (Sp1) and treated with either the isotype control (IgG) oranti-IL-20β antibody. Lungs were collected at day 3 after infection.

FIG. 8: Treatment with IL-20 cytokines and anti-IL-20β antibodymodulated in a different manner the ability of monocyte-deriveddendritic cell (MDDC) to prime IL-17 and IL-22 production by T cells inresponse to activation by S. pneumoniae (Sp1).

EXAMPLE

Introduction:

Bacterial complications are a common feature during a wide variety oflung inflammatory disorders such as COPD. In patients with COPD, acuteexacerbation is mostly associated with bacterial infections frequentlydue to Haemophilus influenzae and Streptococcus pneumoniae. Theseinfections provoked a strong inflammatory reaction characterized byneutrophil recruitment, increased production of pro-inflammatorycytokines and accelerated the progression of the disease. Appropriatemodels are needed to better define mechanisms responsible for bacterialsusceptibility during the exacerbation of COPD. We have alreadydeveloped a murine model of COPD by chronic exposure to cigarette smoke(CS) during 12 weeks. This exposure strongly modifies the innate immuneresponse in the lung and the activation of invariant natural killer T(iNKT) cells (Pichavant M et al., Mucosal Immunol, 2014). To betterdefine the mechanisms responsible for bacterial susceptibility, we havenow developed experimental models of COPD exacerbation in C57/BL6 micechronically exposed to CS and then, infected by the local administrationof sub-lethal doses of H. influenzae and S. pneumoniae.

IL-17 and IL-22 Response to Bacterial Infection is Altered in COPD Mice:

Infected COPD mice develop a strong lung infection with SP (associatedwith an increased inflammatory reaction) whereas naïve mice are able toclear the bacteria within 24 hours. This defect in bacterial clearanceis associated with a lower production of both IL-17 and IL-22 in the BALand after restimulation of lung cells. The defect in IL-17 and IL-22 isrelated to a decreased percentage of NK, NKT cells as well as innatelymphoid cells (ILC) positive for these cytokines in the lung ofinfected COPD mice as compared to infected air-exposed mice. Moreover,the supplementation with recombinant IL-22 allows to accelerate theclearance of the bacteria and to limit the consequences of the infectionby S. pneumoniae in COPD mice. In COPD mice, infection with H.influenzae is associated with a defect in the production of IL-22 whichalso involved the same cells as observed for SP (ILC, NK and NKT cells).Since the role of IL-22 is unknown during infection by H. influenzae, weobserved that IL-22−/− mice reproduce a phenotype close to that of COPDmice (increased susceptibility, higher inflammatory response and lungremodeling). These data show that COPD mice are more susceptible toinfection by H. influenzae and S. pneumoniae than control mice and adeficient production of IL-17 and/or IL-22 may favor the development ofbacteria-induced COPD exacerbations.

IL-17 and IL-22 Response to Bacterial Infection is Altered inMononuclear Cells from COPD Patients:

Although a defect in the production of IL-17 and IL-22 might play a rolein the susceptibility to bacterial infection during COPD, there is nodata reporting the concentrations of these cytokines in the lung frompatients with AE-COPD. In order to estimate the capacity of COPDpatients to produce Th17 cytokines in response to bacteria, we firstanalyze the response to S. pneumoniae of blood mononuclear cells (MNC)from COPD patients in comparison with healthy smokers and not smokers. Apositive control of MNC activation was also included by addition ofphytohaemagglutinin (PHA). The concentrations of cytokines inunstimulated cells were not significantly different among the 3 groups(FIG. 1). Whereas both stimuli significantly increased the levels ofIL-17 and IL-22 in not smokers (controls) and smokers, the exposure toSp did not significantly amplify the secretion of these cytokines inCOPD patients. The response to PHA was also partially altered in COPDpatients, mainly for IL-17 and IL-22. In order to identify the cellsources for these cytokines in response to S. pneumoniae, we analysedthe intracellular staining for IL-17 and IL-22 in these MNC. As comparedwith smokers and not smokers, the production of IL-17 and IL-22 wasaltered in Lin− (potentially the ILC), iNKT and NK cells but not in Tγδcells from COPD patients (data not shown).

Production of IL-20 Cytokines after Exposure to Cigarette Smoke and inResponse to Bacterial Infection:

Since IL-20 cytokines are related to IL-22 and their production isinduced by some bacteria (Staphylococcus Aureus), we evaluated theexpression of these cytokines in our experimental models. After chronicexposure to CS, the expression of IL-19 and IL-20 mRNA was increased inthe lung of COPD mice as compared to controls (air-exposed animals)(FIG. 2). In addition, air mice infected with S. pneumoniae did notproduce IL-19 and IL-24 whereas IL-20 mRNA expression was upregulated.In COPD mice, infection with S. pneumoniae markedly enhanced theexpression of IL-19, IL-20 and IL-24 even we compared the results to thenot-infected COPD mice.

Similar results were obtained after infection with not typeable H.influenzae (NTHI), namely, an additive effect of CS exposure andbacterial infection on the expression of these cytokines.

We also analyzed the expression of these cytokines in human dendriticcells (DC) and airway epithelial cells (AEC) activated in vitro by CSextract (CSE) and/or bacteria. As reported in FIG. 3, infection with SP1did not significantly increased the expression of IL-19 and IL-24. Incontrast, exposure to CSE had a strong effect on both cytokines alone orin presence of S. pneumoniae. Similar results were obtained withmonocyte-derived DC (data not shown).

Function of IL-20 Cytokines in Response to Bacterial Infection DuringCOPD:

Since Myles I A et al (Nat Immunol. 2013) reported that signaling viathe IL-20 receptor inhibits the production of IL-17 and IL-22 to promotecutaneous infection, we hypothesized that these receptors wereimplicated in the defect in IL-17 and IL-22 response observed inbacterial infection of COPD mice. To demonstrate this, we used aneutralizing antibody that recognize the IL-20β subunit. This receptoris common to both receptors binding IL-19, IL-20 or IL-24(IL-20Rα/IL-20Rβ, IL-22Rα/IL-20Rβ). This commercially available antibodyanti-IL-20β (clone 20RNTC, which recognized both human and mouse) isintraperitoneally administrated one day before the infection and the dayafter (50 μg/injection/mouse). For these preliminary experiments, weanalyzed the bacterial load and the expression of Th17 cytokines.

As illustrated in FIG. 4, treatment with blocking anti-IL-20β antibodiesstrongly decreased the bacterial load in the BAL and the lung tissueafter infection with S. pneumoniae. Similar results were obtained afterinfection with H. influenzae (data not shown). In contrast, thistreatment had a moderate or no effect in Air infected mice suggestingthat the activity of this antibody is related to the expression level ofthe IL-20R ligands.

In parallel, we evaluated the expression of Th17 cytokines in the lungsof infected COPD mice. As illustrated in FIG. 5, an increased secretionof IL-22 was detected in lung cells from COPD mice treated withanti-IL-20Rb antibody as compared to the mice receiving the isotypecontrol, whatever at day 1 and 2 after infection with NTHI.

CONCLUSION

Altogether, these data underlines the interest of antagonist of IL20cytokines (e.g. blocking anti-IL-20β antibodies) to limit thesusceptibility to infection in COPD mice potentially through anincreased production of IL-22.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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1. A method of treating acute exacerbation of chronic obstructivepulmonary disease in a subject in need thereof comprising administeringthe subject with a therapeutically effective amount of an antagonist ofIL-20 cytokines.
 2. The method of claim 1 wherein the acute exacerbationof COPD is caused by a bacterial infection, by a viral infection or byair pollution.
 3. The method of claim 2 wherein the bacterial infectionis due to Streptococcus pneumoniae, Haemophilus influenzae, or Moraxellacatarrhalis.
 4. The method of claim 1 wherein the subject experienced anacute exacerbation of COPD or is at risk of experiencing an acuteexacerbation of COPD.
 5. The method of claim 1 wherein the subject is afrequent exacerbator.
 6. The method of claim 1 wherein the treatment isa prophylactic treatment.
 7. The method of claim 1 wherein theantagonist of IL-20 cytokines is delivered to the respiratory tract. 8.The method of claim 1 wherein the antagonist of IL-20 cytokines isadministered to the subject in combination with an antiviral agent or ananti-bacterial agent.
 9. The method of claim 1 wherein the antagonist ofIL-20 cytokines is an antibody directed against a IL-20 cytokine, ananti-sense nucleic acid molecule directed to an IL-20 cytokine, a smallinterfering RNA (siRNA) directed toward an nucleic acid encoding for aIL-20 cytokine, a microRNA directed toward a nucleic acid encoding for aIL-20 cytokine, an anti-antibody directed against a receptor of a IL-20cytokine, an antisense nucleic acid molecule directed to a subunit of areceptor for a IL-20 cytokine, an siRNA or a microRNA directed to anucleic acid encoding for a subunit of a receptor for a IL-20 cytokine.10. The method of claim 1 wherein the antagonist of IL-20 cytokines isan antibody.
 11. The method of claim 1 wherein the antagonist of IL-20cytokines is selected from the group consisting of anti-IL-19antibodies, anti-IL-20 antibodies, anti-IL-24 antibodies, anti-IL20R1antibodies, and anti-IL20R2 antibodies.
 12. The method of claim 1wherein the antagonist of IL-20 cytokines is a polypeptide whichcomprises all or a portion of the extracellular domains of an IL-20receptor.
 13. The method of claim 1 wherein the antagonist of IL-20cytokines is a polypeptide which comprises all or a portion of theextracellular domains of an IL-20 receptor which is fused to animmunoglobulin constant domain.
 14. The method of claim 1 wherein theantagonist of IL-20 cytokines comprises the extracellular domain of theIL-20R1 polypeptide and the extracellular domain of the IL-20RII whichare covalently linked together.
 15. The method of claim 14 wherein oneextracellular domain has a constant region of a heavy chain of animmunoglobulin fused to its carboxy terminus and the other extracellulardomain has a constant light chain of an immunoglobulin fused to itscarboxy terminus such that the two polypeptides come together to form asoluble receptor and a disulfide bond is formed between the heavy andthe light immunoglobulin chains.